Trypanosoma cruzi is a blood and tissue parasite that affects millions of individuals causing significant human morbidity and mortality. T. cruzi must attach to and enter mammalian cells in order to complete its intracellulal development and disseminate infection in the body. Understanding the molecular mechanisms of T. cruzi infection will lead to new approaches in its treatment and control. In addition, basic understanding of the role of cellular genes in T. cruzi pathogenesis may provide new targets for therapy of known as well as emerging infectious agents. Our long-range goal is to understand the molecular mechanisms that allow T. cruzi to infect mammalian cells, so that specific molecular intervention strategies can be developed against T. cruzi infection. The objective of this application is to identify which host cellular genes control mammalian cell infection by T. cruzi. The hypothesis of this application is that a set of host cellular genes is responsible for successful establishment of T. cruzi infection in mammalian cells. We have formulated this hypothesis based on strong preliminary results, which identified three host genes that may control selected steps of T. cruzi infection. Furthermore, supporting this hypothesis is the fact that we have selected additional mutant cell clones, with tagged genomic sequences, which are resistant to trypanosome infection. These clones will facilitate the identification of new host genes required for cellular infection by T.cruzi. We have recently developed a new strategy to identify host cellular genes required for T. cruzi infection. This novel approach uses insertional mutagenesis with the U-3 retrovirus entrapment vector to mutate individual host cell genes and then select mutant cell clones resistant to infection with T. cruzi. We will test our hypothesis by pursuing the following specific aims: (i) Identify host genes involved in cellular infection by T. cruzi, and (ii) determine the roles of the identified host genes in the cellular steps of T. cruzi infection. Mutagenized cell clones that are resistant to T. cruzi infection will be characterized for the gene disrupted by the gene-trap retrovirus event by established molecular biologic techniques. Once the genomic sequence of the disrupted gene is determined, the expression of the gene will be determined. The function of isolated genes in the T. cruzi cycle of infection will be determined by assessing binding of trypanosomes, entry, differentiation, multiplication and parasites released by the mutant cells and in stable transfected mutant cells with cDNAs corresponding to specific interrupted genes. Preliminary sequence data of three disrupted genes suggest that a new receptor for T. cruzi is used by the parasite to bind cells, and a known gene and a novel gene are apparently involved in trypanosome entry.